Microfluidization of radioactive calcium/oxyanion-containing particles

ABSTRACT

Methods of preparing solid apatite particles using a microfluidizer, for use in medical diagnostic imaging such as magnetic resonance imaging, X-ray, and ultrasound. The desired apatite particles are synthesized, passed through a microfluidizer, and purified to remove excess base, salts, and other materials used to synthesize the particles. The microfluidizer causes two high pressure streams to interact at ultra high velocities in a precisely defined microchannel. Microfluidization of preparations causes small (&lt;5 μm) and uniform particles to be formed. Coating and purifying (especially by tangential flow filtration) the particles improves particle stability.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 08/249,776 filed on Jul.26, 1994; now U.S. Pat. No. 5,419,892 which is a divisional applicationof U.S. Ser. No. 08/038,329 filed on Mar. 29, 1993 issued as U.S. Pat.No. 5,342,609; which is a Continuation-In-Part application of U.S. Ser.No. 07/948,540 filed on Sep. 22, 1992 issued as U.S. Pat. No. 5,344,640;which is a Continuation-In-Part application of U.S. Ser. No. 07/784,325filed on Oct. 22, 1991 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the preparation of calcium/oxyanion-containingparticles for use in medical diagnostic imaging, such as magneticresonance imaging ("MRI"), ultrasound, and X-ray. In particular, thepresent invention is directed to the use of a microfluidizer for thepreparation of calcium/oxyanion-containing particles having a uniformsmall (<5 μm) size distribution. The present invention also includes theuse of tangential flow filtration for particle purification.

The use of contrast agents in diagnostic medicine is rapidly growing. InX-ray diagnostics, for example, increased contrast of internal organs,such as the kidneys, the urinary tract, the digestive tract, thevascular system of the heart (angiography), etc., is obtained byadministering a contrast agent which is substantially radiopaque. Inconventional proton MRI diagnostics, increased contrast of internalorgans and tissues may be obtained by administering compositionscontaining paramagnetic metal species which increase the relaxivity ofsurrounding protons. In ultrasound diagnostics, improved contrast isobtained by administering compositions having acoustic impedancesdifferent than that of blood and other tissues.

Often it is desirable to image or treat a specific organ or tissue.Effective organ- or tissue-specific diagnostic agents accumulate in theorgan or tissue of interest. Copending patent application Ser. No.07/948,540, filed Sep. 22, 1992, titled "Treated Apatite Particles forMedical Diagnostic Imaging," which is incorporated herein by reference,discloses the preparation and use of apatite particles for medicaldiagnostic imaging. This patent application also describes methods forpreparing apatite particles which provide organ- or tissue-specificcontrast. By carefully controlling the particle size and route ofadministration, organ specific imaging of the liver, spleen,gastrointestinal tract, or blood pool is obtained.

In general, the apatite particles are prepared by modifying conventionalmethods for preparing hydroxyapatite (sometimes referred to as"hydroxylapatite"). For example, stoichiometric hydroxyapatite, Ca₁₀(OH)₂ (PO₄)₆, is prepared by adding an ammonium phosphate solution to asolution of calcium/ammonium hydroxide. Useful apatite particles mayalso be prepared by replacing calcium with paramagnetic metal ions.Other apatite derivatives are prepared by replacing the OH⁻⁻ with simpleanions, including F⁻⁻, Br⁻⁻, I⁻⁻, or 1/2[CO₃ ²⁻⁻ ].

Various techniques for controlling the particle size for certain calciumphosphate-containing compounds (apatites) are disclosed in copendingapplication Ser. No. 07/948,540. For example, slower addition rates(introduction of the precipitating anion or cation), faster stirring,higher reaction temperatures, and lower concentrations generally resultin smaller particles. In addition, sonication during precipitation,turbulent flow or impingement mixers, homogenization, and pHmodification may be used to control particle size. Other means, such ascomputer controlled autoburets, peristaltic pumps, and syringes, may beused to control the release of precipitating ions to produce smallerparticles.

Due to the small size and nature of apatite particles, they tend toaggregate. Particle aggregation may be inhibited by coating theparticles with coating agents, while agglomerated particles may bedisrupted by mechanical or chemical means and then coated with a coatingagent having an affinity for the apatite.

One preferred method of obtaining small, uniformly sized,manganese-doped apatite particles is to dropwise add a degassed solutionof (NH₄)₂ HPO₄ and NH₄ OH into a rapidly stirring degassed solution ofCa (NO₃)₂ •4H₂ O and Mn (NO₃)₂ •6H₂ O. The resulting apatite particlesare then reacted with a solution of 1-hydroxyethane-1,1-diphosphonicacid (HEDP). The smaller particles are separated from larger particlesby repeated centrifuging and collection of the supernatant. Theparticles are then washed to remove base and salts by centrifuging at ahigher rpm, discarding the supernatant, resuspending the solid pellet inwater, and recentrifuging.

Although the foregoing procedure produces small-sized apatite particleshaving good size distribution and good medical diagnostic imagingproperties, the repeated centrifuging, decanting, and washing causes theprocess to be tedious and time-consuming. It, therefore, would be asignificant advancement in the art to provide an improved method forrapidly preparing calcium/oxyanion-containing particles for medicaldiagnostic applications having a controlled particle size distributionand good yield.

Such methods for preparing calcium/oxyanion-containing particles aredisclosed and claimed herein.

SUMMARY OF THE INVENTION

The present invention provides methods of preparingcalcium/oxyanion-containing particles, including apatites and apatiteprecursors, using a microfluidizer. The particles thus prepared, are foruse in medical diagnostic imaging, such as magnetic resonance imaging,X-ray, and ultrasound applications. The desiredcalcium/oxyanion-containing particles are synthesized, passed through amicrofluidizer, and purified to remove excess base, salts, and othermaterials used to synthesize the particles. The microfluidizer causestwo high pressure streams to interact at ultra high velocities in aprecisely defined microchannel. Use of the microfluidizer results insignificant reduction in the average particle size. Purifying theparticles, preferably using tangential flow filtration, as well ascoating the particles, improves particle stability.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graphical representation of the particle size distributionof manganese-doped hydroxyapatite particles prepared according toExample 8, before and after passing through a microfluidizer.

FIG. 2 is a graphical representation of the osmolality of a particulatesuspension after sequential passes through a tangential flow filtrationsystem as described in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

The present, invention provides methods for preparingcalcium/oxyanion-containing particles, including apatites and apatiteprecursors, especially hydroxyapatite, having uniform, small (<5 μm)particle size and uniform distribution through use of a microfluidizer.

As used herein, calcium/oxyanion-containing particles include calciumphosphate minerals, apatites, and apatite precursors of the generalformula Ca_(n) M_(m) X_(r) Y_(s), where M is a paramagnetic metal ion,radiopaque metal ion, radioactive metal ion, or stoichiometric mixtureof metal ions, X is a simple anion, Y is an oxyanion includingtetrahedral oxyanions, protonated or unprotonated, carbonate, ormixtures thereof, m is from 0 to 10, n is from 1 to 10, s is >1, and ris adjusted as needed to provide charge neutrality.

As used herein, apatite precursors include compounds within the scope ofthe above general formula having one or more amorphous phases which,when sintered, may become crystalline apatites.

Possible paramagnetic metal ions which can be used in thecalcium/oxyanion-containing particles of the present invention include:chromium(III) , manganese(II) , iron(II), iron(III), praseodymium(III),neodymium(III), samarium(III), ytterbium(III), gadolinium(III),terbium(III), dysprosium(III), holmium(III), erbium(III), or mixtures ofthese with each other or with alkali or alkaline earth metals.

Certain radiopaque heavy metals, such as bismuth, tungsten, tantalum,hafnium, lanthanum and the lanthanides, barium, molybdemnn, niobium,zirconium, and strontium may also be incorporated into particles toprovide X-ray contrast.

Typical simple anions which can be used in thecalcium/oxyanion-containing particles of the present invention include:OH⁻⁻, F⁻⁻, Br⁻⁻ I⁻⁻, 1/2[CO₃ ²⁻⁻ ], or mixtures thereof. The tetrahedraloxyanions used in the present invention may optionally includeradiopaque metals or radioactive metals. Suitable tetrahedral oxyanionsare nonoxidizing and stable to hydrolysis. Examples of suitabletetrahedral oxyanions for use in the present invention include: PO₄ ³⁻⁻,AsO₄ ³⁻⁻, WO₄ ²⁻⁻, MoO₄ ²⁻⁻, VO₄ ³⁻⁻, Sio₄ ⁴⁻⁻, and GeO₄ ⁴⁻⁻, and whenstable protonated forms of these. Phosphate is a currently preferredtetrahedral oxyanion.

By controlling the particle size, organ specific imaging or therapy ofthe liver or gastrointestinal tract is obtained. When apatite particleshaving a size in the range from about 5 nm to about 5 μm are injectedinto the vascular system, the particles collect in the liver or spleen(the RES system) because a normal function of these organs is to purifythe blood of foreign particles. Once the particles have collected in theliver or spleen, these organs may be imaged by the desired medicaldiagnostic imaging technique.

Depending on the diagnostic imaging technique, calcium/oxyanioncontaining particles are treated to be paramagnetic, radiopaque, orechogenic. For example, paramagnetic metal species may be incorporatedinto the particles to improve magnetic resonance contrast, andradiopaque species may be incorporated to provide X-ray contrast.Particle density, and corresponding echogenic characteristics, can becontrolled to impart low or high acoustic impedance relative to blood.The calcium/oxyanion-containing particles may also be fluorinated toform stable, nontoxic compositions useful for ¹⁹ F imaging. The presenceof a paramagnetic metal species in these particles may reduce ¹⁹ F andproton relaxivity, thereby enhancing MRI, MRS, or MRSI.

Hydroxyapatite doped with a paramagnetic metal can be prepared by mixinga basic (pH 10-12) phosphate solution with a calcium/paramagnetic metalsolution at native pH. It has been found that the paramagnetic ionsincorporated into the apatite particle tend to oxidize during particlesynthesis. To prevent metal oxidation the amount of oxygen in theaqueous reactant solutions is minimized. Oxygen minimization is obtainedby synthesis at high temperature, such as 100° C. or by degassing theaqueous reactant solutions with an inert gas such as argon, nitrogen, orhelium.

Antioxidants, such as gentisic acid and ascorbic acid, added during orafter apatite particle synthesis may also be used to prevent metal ionoxidation. Reducing agents, such as NaBH₄, have been found to reducemetal ions that are unintentionally oxidized during apatite particlesynthesis.

Paramagnetic particles may also be prepared by adsorbing paramagneticmetal ions onto the particle. For example, manganese can be adsorbed tohydroxyapatite particles by taking a slurry of hydroxyapatite and addingMn(NO₃)₂ with stirring. Applying energy, such as ultrasonic power orheat, to the resulting mixture may also facilitate the reaction. Theresulting mixture can be separated by either centrifugation anddecantation or by filtration. Any excess manganese may be removed bywashing with large amounts of water. The manganese adsorbed particlescan then be stabilized against oxidation and particle agglomeration witha suitable coating agent. The same procedure may be used with otherparamagnetic cations. The amount of manganese adsorbed onto the particlesurface, as a percentage of the total calcium in the particle, is in therange from about 0.1% to about 50%. Such particles exhibit very highrelaxivities and rapid liver enhancement in magnetic resonance imagingstudies.

Particle Size Reduction and Production of Particles of Uniform Sizeusing a Microfluidizer

It has been found that passing calcium/oxyanion-containing particles,including apatites and apatite precursors, through a microfluidizerresults in dramatic particle size reduction. A microfluidizer, such asthose produced by Microfluidics Corporation, Newton, Mass., causes twohigh pressure fluid streams to interact at ultra high velocity. It ispostulated that shear, impact and cavitation forces act on the fluidstreams to achieve submicron particle reduction with uniformdistribution. Fluid pressures typically range from 2000 psi to 30,000psi with some production size microfluidizers capable of handlingpressures up to 40,000 psi.

Experimental results suggest that particle size reduction using amicrofluidizer can be obtained from apatite particles regardless ofwhether the particles are first stabilized with a coating agent orpurified from the base, salts, and other compounds used to prepare theparticles. The particles may be purified or unpurified, coated oruncoated when passed through the microfluidizer. However, it appearsthat the microfluidized apatite particles show better stability withremoval of the base, salts, and other compounds in the reaction mixture.The particles tend to become larger when stored in the basic reactionsolution, but growth of purified particles is either stopped orinhibited by purification of the particles from the mixture. Particlepurification can be obtained by processes such as repeated centrifugingand decanting, passing through a desalting column, and filtration,preferably tangential flow filtration or ultrafiltration.

Particle Coating

Stabilized calcium/oxyanion-containing particles, including apatites andapatite precursors, are desirable for in vivo use as medical diagnosticimaging agents. Such particles tend to aggregate. Although the reasonscalcium/oxyanion-containing particles aggregate is not fully understood,it has been found that several different coating agents are able toinhibit particle aggregation. For example, these particles may bestabilized by treatment with coating agents such as di- andpolyphosphonate-containing compounds or their salts, such as1-hydroxyethane-1,1-diphosphonate (HEDP), pyrophosphate,aminophosphonates; carboxylates and polycarboxylate-containing compoundssuch as oxalates and citrates; alcohols and polyalcohol-containingcompounds; compounds containing one or more phosphate, sulfate, orsulfonate moiety; and biomolecules such as peptides, proteins,antibodies, and lipids all have been shown to inhibit particleaggregation. Such coating agents stabilize the small particles byreducing further particle growth and promoting particle suspension.

When used in magnetic resonance imaging, particle relaxivity is enhancedby allowing more water accessible to the particle surface. By limitingparticle size and increasing the available surface area, relaxivity maybe improved.

In addition to the coating agents identified above, conventionalparticle coating techniques may also be used in the manufacturingprocesses of the present invention. Typical coating techniques areidentified in International Publication Numbers WO 85/02772, WO91/02811, and European Publication Number EP 0343934, which areincorporated by reference.

For instance, agglomerated particles may be disrupted by mechanical orchemical means and then coated with polymers such as carbohydrates,proteins, and synthetic polymers. Dextran having a molecular weight inthe range from about 10,000 to about 40,000 is one currently preferredcoating material. Albumin and surfactants, such as tween 80, have alsobeen used to reduce particle aggregation. One common characteristic ofuseful apatite coating agents is their ability to modify the particlesurface charge, or zeta potential.

It will be appreciated that the calcium phosphate-containing particleswithin the scope of the present invention may be coated before, during,or after passage through the microfluidizer. When coated during passagethrough the microfluidizer, one fluid stream is the coating agent, whilethe other fluid stream is the particulate stream.

The currently preferred mechanical means for reducing particle size ismicrofluidization, but other means such as heating, sonication, otherforms of particle energization, such as irradiation, and chemical means,such as pH modification or combinations of these types of treatment,such as pH modification combined with sonication may be used.

Diagnostic Pharmaceutical Formulations

The calcium/oxyanion-containing particles of this invention may beformulated into diagnostic compositions for parenteral administration.For example, parenteral formulations advantageously contain a sterileaqueous solution or suspension of treated apatite or apatite precursorparticles according to this invention. Various techniques for preparingsuitable pharmaceutical solutions and suspensions are known in the art.Such solutions also may contain pharmaceutically acceptable buffers and,optionally, electrolytes such as sodium chloride. Parenteralcompositions may be injected directly or mixed with a large volumeparenteral composition for systemic administration.

The diagnostic compositions of this invention are used in a conventionalmanner in medical diagnostic imaging procedures such as magneticresonance, X-ray, and ultra-sound imaging. The diagnostic compositionsare administered in a sufficient amount to provide adequatevisualization, to a warm-blooded animal either systemically or locallyto an organ or tissues to be imaged, then the animal is subjected to themedical diagnostic procedure. Such doses may vary widely, depending uponthe diagnostic technique employed as well as the organ to be imaged.

The following examples are offered to further illustrate the presentinvention. These examples are intended to be purely exemplary and shouldnot be viewed as a limitation on any claimed embodiment.

EXAMPLE 1 Preparation of Hydroxyapatite Particles Doped with Mn, Treatedwith HEDP, Purified and Passed through Microfluidizer

Manganese containing hydroxyapatite particles were prepared by thefollowing general procedure. A procedure is described for particlescontaining 10% Mn (compared to the total metal content) but otherpercentages are also applicable.

Into a 1 L erlenmeyer flask were placed 10.5 g of (NH₄)₂ HPO₄, 100 mL ofconcentrated NH₄ OH and 350 mL of D.I. water. The mixture was stirredfor two hours with a continuous heavy argon flow (degassing). In aseparate 1 L erlenmeyer flask were placed 28.9 g of Ca(NO₃)₂ •4H₂ O and2.4 g of Mn(NO₃)₂ •6H₂ O 400 mL of D.I. water. The metal nitratesolution was degassed with argon for 2 hours. The phosphate solution wasthen added dropwise to the rapidly stirred metal nitrate mixture overtwo hours with a peristaltic pump. A continuous argon flow wasmaintained throughout the course of the reaction. The reaction mixturewas stirred for an additional two hours after the addition was complete.A solution of 8.3 mL of a 60% solution HEDP (acid form) in 25 mL of D.I.water was degassed for 30 minutes then added in one aliquot to thehydroxyapatite mixture. The resulting slurry was stirred for 15 minutes.

The entire reaction mixture was centrifuged at one time at 2400 rpm for15 minutes. The supernatant was discarded and the solid residue in eachtube resuspended in water. The slurry was re-centrifuged at 2400 rpm andthe milky supernatant was collected. The solid was resuspended twicemore and centrifuged at 2400 rpm. The three washes were combined andcentrifuged at 7000 rpm for 30 minutes. The resulting solid pellet wasseparated from the supernatant by decantation, and the pellet was washed(D.I. H₂ O) and centrifuged three times, and the supernatants werediscarded. After washing, the solid pellet was suspended in 30 mL ofD.I. H₂ O.

The preparation was stored at room temperature for one month. Theparticle size was analyzed and found to be 280 nm (2.9 chi squared, 0.31coefficient of variance). The particulate suspension was passed througha microfluidizer at approximately 5000 psi. After one pass through themicrofluidizer, the particle size was reduced to 125 nm (0.43 chisquared, 0.35 coefficient of variance). After another pass through themicrofluidizer at a pressure of approximately 10,000 psi, the size didnot change significantly, 144 nm (0.20 chi squared, 0.28 coefficient ofvariance). At three hours and 36 hours after passing through themicrofluidizer, the particle size remained essentially constant at 159nm and 148 nm, respectively.

EXAMPLE 2 Preparation of Hydroxyapatite Particles Doped with Mn andTreated with HEDP and Passed through Microfluidizer Unpurified

Manganese containing hydroxyapatite particles were prepared according tothe procedure of Example 1, except that the particles were not purifiedby centrifuging, decanting, and washing, but left in the base and saltsolution. The particulate suspension (average size >1 μm, chisquared >201) was passed through a microfluidizer at approximately 5000psi. After one pass through the microfluidizer, the particle size was 87nm (2.3 chi squared, 0.41 coefficient of variance). After five passesthrough the microfluidizer at pressures from 5000 psi to 7000 psi, theparticle size was 89 nm (0.88 chi squared, 0.37 coefficient ofvariance).

The resulting particles were too small to pellet at 2400 rpm and wereleft in the base and salts. There was no indication that multiple passesthrough the microfluidizer made smaller particles, but it appears theuniformity was increased. Twenty hours after passing through themicrofluidizer the particle size has increased to 713 nm (21.1 chisquared, 0.53 coefficient of variance). Although the chi squared waslarge, indicating a poor fit to a gaussian distribution, the coefficientof variance was small with 99% of the particles less than 2 μm and 75%less than 825 nm. The relaxivity (R₁) of these particles 2 hours afterformation was approximately 22 mM⁻¹ s⁻¹.

EXAMPLE 3 Preparation of Hydroxyapatite Particles Doped with Mn andPassed through Microfluidizer Unpurified with a simultaneous coaxialstream of HEDP

Manganese containing hydroxyapatite particles were prepared according tothe procedure of Example 1, except that the particles were not coatedwith HEDP and were not purified by centrifuging, decanting, and washing,but left in the base and salt solution. The particulate suspension waspassed as one stream into a microfluidizer. The other microfluidizerstream consisted of a HEDP solution prepared according to the procedureof Example 1. The two streams passed through the microfluidizer at apressure of 10,000 psi. The resulting particulate suspension had aparticle size of 70 nm (2.4 chi squared, 0.42 coefficient of variance).The particles were not purified from base and salts. Two hours afterformation the particle size was 87 nm (1.8 chi squared, 0.41 coefficientof variance). Thirty-six hours after formation the particle size was 903nm (0.84 chi squared, 0.45 coefficient of variance) indicating theparticles had grown uniformly to a large size. The relaxivity (R₁) ofthese particles was 24 mM⁻¹ s⁻¹.

EXAMPLE 4 Preparation of Hydroxyapatite Particles Doped with Mn andPassed through Microfluidizer Unpurified into Neutral HEDP Solution

Manganese containing hydroxyapatite particles were prepared according tothe procedure of Example 1, except that the particles were not coatedwith HEDP and were not purified by centrifuging, decanting, and washing,but left in the base and salt solution. The particulate suspension waspassed through a microfluidizer at 10,000 psi and into a beaker ofneutral HEDP. The neutral HEDP solution was prepared from 8.3 mL of a60% solution HEDP (neutral form) in 25 mL of D.I. water.

The resulting particulate solution had an average particle size of 1333nm (7.3 chi squared, 0.40 coefficient of variance). Two hours afterformation, the particle size was 884 nm (8.3 chi squared, 0.46coefficient of variance). The results suggest that the use of acidicHEDP is useful in the formation of small particles and the neutral formof HEDP may be used when larger particles are desired.

Examples 1-4 indicate that the particle size of manganese dopedhydroxyapatite may be substantially reduced by the shear, impact andcavitation forces present within the microfluidizer.

EXAMPLE 5 Preparation of Hydroxyapatite Particles Doped with Mn, Washed,Coated with Aminotri(methylene Phosphonic acid) (ATMP), and Passedthrough Microfluidizer

Manganese containing hydroxyapatite particles were prepared according tothe procedure of Example 1, except that the particles were not coatedwith HEDP and the particles were washed free of base and salts bycentrifuging three times at 2400 rpm. Degassed water was used to washthe pelleted particles following centrifuging. An ATMP solution wasprepared by mixing 0.0027 moles or 1.6 mL of a 50% aqueous solution with25 mL D.I. H₂ O and degassing for 30 minutes under argon. The ATMPsolution was added dropwise to the washed particles resulting in a"white" slurry. The slurry was passed through a microfluidizer at 10,000psi. After passing through the microfluidizer, the particles had anestimated size of 84 nm (1.3 chi squared, 0.52 coefficient of variance).There was some oxidation of manganese with time as evident from a brownappearance in the particles. After six days there were two populationsof particles, 46 nm and >2 μm. The percentages of each component couldnot be determined due to the limits of the particle analyzer andsettling of the larger particles.

EXAMPLE 6 Preparation of Hydroxyapatite Particles Doped with Mn, Coatedwith HEDP, Passed through Microfluidizer, and Purified

Manganese containing hydroxyapatite particles were prepared according tothe procedure of Example 1, except that the particles were not coatedwith HEDP and were not purified by centrifuging, decanting, and washing,but left in the base and salt solution. An HEDP solution preparedaccording to the procedure of Example 1 was added dropwise to theparticles. The particle size before passing through a microfluidizer was1498 nm (13.4 chi squared, 0.93 coefficient of variance). After passingthe particulate suspension through the microfluidizer at 10,000 psi. theparticle size was 62 nm (0.27 chi squared, 0.47 coefficient ofvariance). About 2-3 hours after microfluidization, one half of theparticulate suspension was passed through a Sephadex 10(S-10) desaltingcolumn to remove base, salts, and excess ligand. The remainingparticulate suspension was retained as a control. Following S-10purification, the particle size was 78 nm (3.3 chi squared, 0.44coefficient of variance). Six days later, the particle size of the S-10purified sample was 100 nm (0.40 chi squared, 0.38 coefficient ofvariance). After 12 days, the size of the particles that were passedthrough the microfluidizer but were not purified and stored in the basesolution increased to 744 nm (4.22 chi squared, 0.57 coefficient ofvariance). In contrast, after 12 days the S-10 purified fraction had aparticle size of 77 nm (0.65 chi squared, 0.44 coefficient of variance).

EXAMPLE 7 Preparation of Hydroxyapatite Particles Doped with Mn, Coatedwith ATMP, Passed through Microfluidizer, and Purified

Manganese containing hydroxyapatite particles were prepared according tothe procedure of Example 1, except that the particles were not coatedwith HEDP and were not purified by centrifuging, decanting, and washing,but left in the base and salt solution. An ATMP solution was prepared bymixing 0.0027 moles or 1.6 mL of a 50% aqueous solution with 25 mL D.I.H₂ O and degassing for 30 minutes under argon. The ATMP solution wasadded dropwise to the particles. The particle size before passingthrough a microfluidizer was 1465 nm and difficult to analyze due tosettling. After passing the particulate suspension through themicrofluidizer at 10,000 psi the particle size was 85 nm (0.58 chisquared, 0.41 coefficient of variance). The particulate suspension wasdivided into two parts. One part was passed through a Sephadex 10 (S-10) desalting column to remove base, salts, and excess ligand. Theremaining part of the particulate suspension was retained as a control.Following S-10 purification, the particle size was 67 nm (0.25 chisquared, 0.44 coefficient of variance). Six days later, the particlesize of the S-10 purified sample was 131 nm (0.60 chi squared, 0.39coefficient of variance). There were three populations in the S-10fraction: 66 nm (45%), 193 nm (38%) and 665 nm (16%). After 12 days, thefraction that was stored in base solution had a particle size of 515 nm(0.50 chi squared, 0.47 coefficient of variance).

From the foregoing Examples, it appears the apatite particles arestabilized better with removal of the base, salts, and excessphosphonate. The particles tend to grow at a fast rate when stored inthe reaction solution, but growth of purified particles is eitherstopped or inhibited. There seems to be a preference for the formationof smaller particles when the microfluidizer experiments are carried outin the base rather than the washed particles.

EXAMPLE 8 Preparation of Hydroxyapatite Particles Doped with Mn, Coatedwith HEDP, Passed through Microfluidizer, and Purified by TangentialFlow Filtration

Manganese containing hydroxyapatite particles were prepared by thefollowing general procedure. A procedure is described for particlescontaining 10% Mn but other percentages are also applicable.

Into a 1 L erlenmeyer flask were placed 10.55 g of (NH₄)₂ HPO₄, 100 mLof concentrated NH₄ OH and 300 mL of D.I. water. The mixture was stirredfor one hour with a continuous heavy argon flow (degassing). In aseparate 1 L erlenmeyer flask were placed 28.9 g of Ca(NO₃)₂ •4H₂ O and2.42 g (0.01355 moles) of Mn(NO₃)₂ •6H₂ O in 200 mL of D.I. water. Themetal nitrate solution was degassed with argon for one hour. Thephosphate solution was then added dropwise to the rapidly stirred metalnitrate mixture over 15 minutes with a peristaltic pump. A continuousargon flow was maintained throughout the course of the reaction. Thereaction mixture was stirred for an additional one hour after theaddition was complete. A solution of 5 g or 8.3 mL of a 60% solutionHEDP (acid form) in 20 mL of D.I. water was degassed for 30 minutes thenadded dropwise to the hydroxyapatite mixture. The resulting slurry wasstirred for 1.5 hours.

Two thirds of the reaction mixture was passed through a microfluidizerat 10,000 psi. The particle size before passing through a microfluidizerwas 800 nm (27 chi squared, 0.92 coefficient of variance). After passingthe particulate suspension through the microfluidizer, the particle sizewas 53 nm (2.2 chi squared, 0.48 coefficient of variance). Theparticulate suspension was then purified to remove base, salts, andexcess ligand by passing it through a tangential flow filtration(sometimes referred to as "ultrafiltration") system. The tangential flowfiltration system was obtained from Koch Membrane Systems, Inc.,Wilmington, Mass. After each filtration pass,, the osmolality wasmeasured. A total of 10 filtration passes were made followed by a 3-foldconcentration step.

Following filtration, the particle size was 67 nm (0.43 chi squared,0.44 coefficient of variance). After 12 days, the size of the particlesthat were passed through the microfluidizer but were not purified andstored in the base solution increased to 744 nm (4.22 chi squared, 0.57coefficient of variance). In contrast, after 12 days the filteredfraction had a particle size of 82 nm (2.7 chi squared, 0.41 coefficientof variance).

The results of this Example are illustrated graphically in FIGS. 1 and2.

EXAMPLE 9 Preparation at 100° C. of Hydroxyapatite Particles Modified bySurface-Adsorbed Mn, Coating with HEDP, Passage through Microfluidizer,and Purification

Calcium hydroxyapatite particles are prepared by the followingprocedure:

A solution containing 6.5 g of (NH₄)₂ HPO₄ in 120 mL of D.I. water istreated with 60 mL of concentrated NH₄ OH followed by 90 mL of D.I.water. The resulting solution is stirred for 3 hours at roomtemperature.

Into a 3-neck 1 L round bottom flask equipped with a water cooled andlow temperature condenser sequence (dry ice/isopropanol), mechanicalstirrer and rubber septum are placed 19.4 g of Ca(NO₃)₂ •4H₂ O in 468 mLof D.I. water. The solution is heated to reflux. The phosphate mixtureis added to the rapidly stirred calcium nitrate solution dropwise with aperistaltic pump over one hour. The heat is removed when the addition iscomplete and the reaction mixture is cooled to room temperature. Thehydroxylapatite slurry is stirred overnight at room temperature.

The pH of the reaction mixture is decreased from 9.53 to 8.50 with 169ml of 1 N HCl. Manganese nitrate, Mn(NO₃)₂ •6H₂ O (2.10 g) is added tothe hydroxyapatite mixture and stirred for 1 hour and 15 minutes. Thecolor of the slurry is pale tan. The mixture is passed through atangential flow filter to remove excess manganese nitrate from theapatite particles. The particulate slurry is then treated with 0.54 MHEDP (Ca/HEDP mole ratio=1.2) and stirred for 1.5 hours. The color ofthe mixture is pale pink/purple.

The HEDP treated hydroxyapatite particulate suspension is passed througha microfluidizer at a pressure of 5000 psi. The particulate suspensionis then purified to remove base, salts, and excess ligand by passing itthrough a tangential flow filtration system.

EXAMPLE 10 Preparation of Mn-Doped Hydroxyapatite Particles Having aFunctionalized Coating Agent, Passage Through Microfluidizer andPurification by Filtration

This example describes the general preparation of hydroxyapatiteparticles having a functionalized coating agent where the functionalizedcoating agent is defined as one with the ability to bind tightly to theparticles and contains a pendant group to which other organicbiomolecules or organic may be attached. The particles are prepared byadding 0.1 to 100 mole % of an appropriate coating agent to a slurry ofMn(II) substituted hydroxyapatite with 0.1 to 100 mole % Mn based on theCa used in the reaction. The mixture is stirred from 1 to 360 minutes attemperatures in the range from 4° C. to 100° C. The particulatesuspension is passed through a microfluidizer at a pressure in the rangefrom 2000 to 20,000 psi, and the solid separated from the supernatantand purified from excess ions and coating agent by tangential flowfiltration. The solid may be treated with a metal salt (0.01 to 10 mole% based on the total metal in the preparation). This is especiallyappropriate if the coating agent contains a pendant chelating groupdesigned to capture and hold tightly the metal when subjected to invitro and/or in vivo solutions. The resultant solid is purified toremove loosely attached coating agent or free metal/coating agentcomplex by tangential flow filtration.

EXAMPLE 11 Preparation of Hydroxyapatite Particles by treating withDiethylenetriamine-penta(methylenephosphonic acid), Surface AdsorbingMn, Passing through Microfluidizer, and Purification

Calcium hydroxyapatite is prepared by the following procedure andtreated with the polyphosphonate,diethylene-triaminepenta(methylenephosphonic acid) (abbreviatedDETAPMDP) having the following formula: ##STR1##

A basic ammonium phosphate solution is prepared using 6.34 g of (NH₄)₂HPO₄ in 120 mL of D.I. water. Concentrated ammonium hydroxide (60 mL) isadded followed by 90 ml of D.I. water. The mixture is stirred for 4hours at room temperature.

A solution of 19.0 g of Ca(NO₃)₂ •4H₂ O in 468 mL of D.I. water isplaced in a 3-neck 1 L round bottom flask. The reaction setup includes amechanical stirrer, water cooled and low temperature (dryice/isopropanol) condenser arrangement, and a rubber septum. Thesolution is heated to reflux with rapid stirring. The basic phosphatesolution is added dropwise with a peristaltic pump over one hour. Theheat is removed after the addition is complete and the reaction mixturestirred overnight at room temperature.

The hydroxyapatite slurry is treated with a solution of DETAPMDP(Ca/DETAPMDP mole ratio=1.1, pH of DETAPMDP 6.3 ) and stirred at roomtemperature for 2.5 hours. The phosphonate treated mixture is thenreacted with Mn(NO₃)₂ •6H₂ O (Ca/Mn mole ratio=2.3 ) and stirred for anadditional 3.5 hours. The reaction mixture is passed through amicrofluidizer at a pressure of 5000 psi and purified by tangential flowfiltration.

From the foregoing, it will be appreciated that the present inventionprovides an improved method for preparing solid calciumphosphate-containing particles for medical diagnostic applicationshaving a controlled particle size distribution and good yield.

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changeswhich come within the meaning and range of equivalency of the claims areto be embraced within their scope.

What is claimed is:
 1. A method of preparing acalcium/oxyanion-containing particle for use in medical diagnosticimaging of body organs and tissues comprising the steps of:(a) obtainingcalcium/oxyanion-containing particles having the following generalformula:

    Ca.sub.n M.sub.m X.sub.r Y.sub.s

wherein M is radioactive metal ion or stoichiometric mixture of metalions, X is a simple anion, Y is an oxyanion, tetrahedral oxyanion,protonated or unprotonated, carbonate, or mixtures thereof, n is from 1to 10, m is from 0 to 10, s is ≧1, and r is adjusted as needed toprovide charge neutrality; and (b) passing thecalcium/oxyanion-containing particles through a microfluidizer.
 2. Amethod of preparing a calcium/oxyanion-containing particle for use inmedical diagnostic imaging as defined in claim 1, wherein the particlespassed through the microfluidizer have a particle size in the range fromabout 5 nm to about 5 μm and are used for imaging the liver and spleen.3. A method of preparing a calcium/oxyanion-containing particle for usein medical diagnostic imaging as defined in claim 1, wherein theparticles passed through the microfluidizer have a particle size in therange from about 1 nm to about 50 nm and are used for imaging the bloodpool.
 4. A method of preparing a calcium/oxyanion-containing particlefor use in medical diagnostic imaging as defined in claim 1, furthercomprising the step of coating the particles with a coating agent tostabilize the calcium/oxyanion-containing particles.
 5. A method ofpreparing a calcium/oxyanion-containing particle for use in medicaldiagnostic imaging as defined in claim 4, wherein the coating agent isselected from aminophosphonates, biomolecules, and compounds containingone or more phosphonate, carboxylate, phosphate, sulfate, or sulfonatemoiety.
 6. A method of preparing a calcium/oxyanion-containing particlefor use in medical diagnostic imaging as defined in claim 4, wherein thecoating agent contains one or more phosphonate moieties.
 7. A method ofpreparing a calcium/oxyanion-containing particle for use in medicaldiagnostic imaging as defined in claim 4, wherein the coating agent is1-hydroxyethane-1,1-diphosphonic acid and physiologically compatiblesalts thereof.
 8. A method of preparing a calcium/oxyanion-containingparticle for use in medical diagnostic imaging as defined in claim 4,wherein the coating agent contains a reactive functional group.
 9. Amethod of preparing a calcium/oxyanion-containing particle for use inmedical diagnostic imaging as defined in claim 8, wherein the reactivefunctional group is an amine, active ester, alcohol, or carboxylatefunctional group.
 10. A method of preparing acalcium/oxyanion-containing particle for use in medical diagnosticimaging as defined in claim 8, wherein the reactive functional group iscapable of chelating a metal ion.
 11. A method of preparing acalcium/oxyanion-containing particle for use in medical diagnosticimaging as defined in claim 4, wherein the step of coating the particleswith a coating agent is performed after the step of passing theparticles through a microfluidizer.
 12. A method of preparing acalcium/oxyanion-containing particle for use in medical diagnosticimaging as defined in claims 4, wherein the step of coating theparticles with a coating agent is performed during the step of passingthe particles through a microfluidizer.
 13. A method of preparing acalcium/oxyanion-containing particle for use in medical diagnosticimaging as defined in claim 4, wherein the step of coating the particleswith a coating agent is performed before the step of passing theparticles through a microfluidizer.
 14. A method of preparing acalcium/oxyanion-containing particle for use in medical diagnosticimaging as defined in claim 1, further comprising the step of purifyingthe calcium/oxyanion-containing particles from base and salts used tosynthesize the calcium/oxyanion-containing particles.
 15. A method ofpreparing a calcium/oxyanion-containing particle for use in medicaldiagnostic imaging as defined in claim 14, wherein thecalcium/oxyanion-containing particles are purified by filtration.
 16. Amethod of preparing a calcium/oxyanion-containing particle for use inmedical diagnostic imaging as defined in claim 14, wherein thecalcium/oxyanion-containing particles are purified by tangential flowfiltration.
 17. A method of preparing a calcium/oxyanion-containingparticle for use in medical diagnostic imaging as defined in claim 14,wherein the calcium/oxyanion-containing particles are purified bypassage through a desalting column.
 18. A method of preparing acalcium/oxyanion-containing particle for use in medical diagnosticimaging as defined in claim 14, wherein the step of purifying thecalcium/oxyanion-containing particles is performed after the step ofpassing the particles through a microfluidizer.
 19. A method ofpreparing a calcium/oxyanion-containing particle for use in medicaldiagnostic imaging as defined in claim 14, wherein the step of purifyingthe calcium/oxyanion-containing particles is performed before the stepof passing the particles through a microfluidizer.
 20. A method ofpreparing a calcium/oxyanion-containing particle for use in medicaldiagnostic imaging as defined in claim 1, wherein the step of obtainingthe calcium/oxyanion-containing particles is performed by passingreaction streams containing base and salts required to synthesize thecalcium/oxyanion-containing particles through a microfluidizer.